Activation of STIM1/Orai1‑mediated SOCE in sepsis‑induced myocardial depression
- Authors:
- Published online on: June 16, 2022 https://doi.org/10.3892/mmr.2022.12775
- Article Number: 259
Abstract
Introduction
Sepsis is life-threatening organ dysfunction caused by an uncontrollable immune response induced by infection (1). Among the multi-organ dysfunction syndromes induced by sepsis, 40% of patients have reduced cardiac function and 20% have cardiac dysfunction (2). Sepsis-induced myocardial depression contributes to the prognosis of patients with sepsis and exploring the pathogenesis of sepsis-induced cardiac depression may alleviate the prognosis of these patients (3). The mechanism of cardiac depression may result from structural effects due to cardiomyocyte death, as well as functional effects caused by cardiac dysfunction. Calcium (Ca2+) homeostasis is essential for normal cardiac excitation contraction coupling (4). Ca2+ also serves an essential role in the signalling pathways associated with cellular termination, including induction of apoptosis and damage to structural proteins (5,6). Lipopolysaccharide (LPS) efficiently increases the intracellular Ca2+ concentration in cardiomyocytes (7) and disruption of Ca2+ homeostasis reduces dystrophin, actin and myosin expression, but increases the expression of calpain. It also leads to decreases in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (8). Moreover, inhibitors of the sarcoplasmic reticulum (SR) Ca-ATPase and Ca2+ chelators decrease cellular Ca2+ levels, which may reduce the cardiac apoptosis induced by LPS (9). However, the transient Ca2+ concentrations responsible for contraction fluctuate several times a minute from 0.1 µM in the diastole period to 1–10 µM in the systole period, which does not appear to activate cellular signalling events (10). Therefore, the activation of signalling pathways by Ca2+ is distinct from the Ca2+ fluctuations responsible for normal cycles of myocardial excitation, contraction and relaxation.
Voltage-gated Ca2+ entry is the main source of elevated intracellular cardiac Ca2+ levels and originates from Ca2+ influx via the plasma membrane (11). Store-operated Ca2+ entry (SOCE) is an additional pathway of Ca2+ entry originally associated with Ca2+ changes in unexcited cells (12–14). Neonatal and embryonic cardiomyocytes depleted of Ca2+ in the SR were previously demonstrated to efficiently induce Ca2+ influx, which was blocked by the SOCE inhibitor SKF-96365, but not the L-type Ca2+ channel (LTCC) blocker verapamil (15). SOCE has also been detected in adult cardiomyocytes using whole-cell patch-clamp experiments (16). However, understanding the role of SOCE in cardiomyocytes was limited until the proteins essential to this signalling pathway were discovered. SOCE is associated with stromal interaction molecule 1 (STIM1) and Orai calcium release-activated calcium modulator 1 (Orai1) (17) and the expression of STIM1 and Orai1 has also been reported in myocardium (18–20). Orai1 is a transmembrane protein of the cytomembrane and is a Ca2+ channel-mediated Ca2+ entry point from the internal environment (21). As a protein located in the cellular membrane, Orai1 does not sense changes in Ca2+ levels in the endoplasmic reticulum (ER) (21). The STIM1 protein is distributed mainly in the ER membrane and can sense changes to the Ca2+ level in the ER (22). It has been demonstrated that depletion of Ca2+ in the ER can induce the clustering and distribution of STIM1 to the ER-plasma membrane junctions, which subsequently mediates the activation of Orai1 (22).
Numerous studies have demonstrated the role of STIM1/Orai1-mediated SOCE in the pathogenesis of cardiac disease (23–26), especially in cardiac hypertrophy (27,28). However, to the best of our knowledge the role of STIM1/Orai1-mediated SOCE in the development of septic myocardial depression has not been reported. During septic myocardial depression, inhibition of the SR Ca-ATPase and enhanced ryanodine receptor leakage induce depletion of Ca2+ in the ER (29). Therefore, we hypothesize that STIM1/Orai1-mediated SOCE contributes to the pathogenesis and development of septic cardiac depression.
Materials and methods
Reagents
LPS and anti-Orai1 antibody (cat. no. SAB3500127) were purchased from Sigma-Aldrich (Merck KGaA). Antibodies for STIM1 (cat. no. ab108994) and Bax (cat. no. ab32503) were purchased from Abcam. Antibody against Bcl-2 (cat. no. 2870T) was purchased from Cell Signaling Technology, Inc. GAPDH (cat. no. BS60630) was purchased from Bioworld Technology, Inc. and secondary antibodies (cat. no. BL003A) for western blotting was purchased from Biosharp Life. TRIzol® reagent was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). QuantiTect Reverse Transcription (RT) Kit and SYBR Green Master Mix were purchased from Takara Biotechnology Co., Ltd. DyLightFluor® 488-conjugated donkey anti-rabbit IgG secondary antibodies (cat. no. BYE026 was obtained from Shanghai Boyun Biotech Co., Ltd. Fluo-3AMacetoxymethyl ester (AM; cat. no. s1056) and bovine serum albumin (BSA) were purchased from Beyotime Institute of Biotechnology. Thapsigargin used for depleting SR Ca2+ stores (TG; cat. no. mb13319) was purchased from Dalian Melone Biotechnology Co., Ltd. and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) kits were purchased from Roche Molecular Diagnostics.
Animals
Male C57BL/6 mice (age, 8 weeks; weight, 20–25 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. and were raised under pathogen-free conditions at 25°C at 50% humidity and 12-h light/dark cycles with free access to food and water. The present study was performed in accordance with the National Institutes of Health Guidelines for Care and Use of Animals (30) and was approved by the Ethics Committee of Wenzhou Medical University (Wenzhou, China).
CLP model
In total, 90 mice were randomly assigned to the following six groups: i) Sham 6 h; ii) Sham 12 h; iii) sham 24 h; iv) CLP 6 h; v) CLP 12 h; and vi) CLP 24 h. The CLP model was established as described previously (31,32). Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally) and following sterilization a midline incision (1 cm) was made to expose the cecum. The cecum was ligated in half and punctured twice using an 18-gauge needle before removing a small amount of stool and placing it in the abdomen. The wound was sutured following the CLP operation. Mice in the sham group underwent a laparotomy without CLP operation. All mice were resuscitated with a subcutaneous injection of a 24 ml/kg sterile saline solution (0.9%). At the corresponding timepoint, mice were sacrificed by cervical dislocation after anaesthesia using pentobarbital sodium (50 mg/kg intraperitoneally). Death was verified by cessation of heartbeat and pupil dilation.
Survival rate
The survival rate of the mice in each group was evaluated within 72 h of the sham or CLP operation. Mice had free access to water and food and were kept under pathogen-free conditions and were monitored every 6 h. All mice were euthanasia at the end of the study.
Evaluation of cardiac function by echocardiography
Cardiac function was assessed via transthoracic echocardiographic examination. Mice were lightly anesthetized with isoflurane anaesthesia (3% induction and 1~2% maintenance). The assessment of left ventricular cardiac function was performed before and after the operation to assess the influence of CLP on cardiac function. Echocardiographic images were obtained using an echocardiography instrument equipped with a 15 MHz phased-array transducer (GE LOGIQ E9; General Electric Company). The motion mode (M-mode) images of the left ventricular dimensions were taken and the LVEF and LVFS were assessed.
Hematoxylin and eosin (H&E) staining
The hearts of the mice were harvested after the sham or CLP operations and fixed in 4% paraformaldehyde at 4°C for 24 h. Paraffin-embedded myocardial tissues were cut into sections (5 µm). H&E staining was performed at room temperature with 0.25% hematoxylin for 10 min and 0.5% eosin for 2 min. Morphological changes were evaluated using the Olympus CKX41SF light microscope (Olympus Corporation).
TUNEL staining
Myocardial apoptosis was detected using the TUNEL method. The heart tissues were fixed in a 4% paraformaldehyde at 4°C for 24 h. Paraffin-embedded myocardial tissues were cut into sections (5 µm) and the slides were stained with 50 µl TUNEL reagent (50 µl terminal deoxynucleotidyl transferase and 450 µl biotin-dUTP) for 60 min at 37°C. Subsequently, the samples were incubated with 50 µl horseradish peroxidase-conjugated streptavidin solution at room temperature for 30 min and were then developed with DAB chromogenic reagent (5 µl 20X DAB + 1 µl 30% H2O2+94 µl PBS) for 5 min at room temperature. The slices were stained with haematoxylin, dehydrated by ethanol and mounted with neutral balata fixation. Representative images were taken using a light microscope. Five random fields were evaluated in each slide.
RT-quantitative (q)PCR
Total RNA was isolated from the myocardium of CLP and sham mice using TRIzol according to the manufacturer's protocol. Complementary DNA was synthesized from 1 µg total RNA using PrimeScript™ RT Master Mix (Takara Biotechnology Co., Ltd) according to the manufacturer's protocol described. qPCR was performed using the SYBR Green Master Mix (Takara Biotechnology Co., Ltd) with the CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc.). The following thermocycling conditions were used: 30 sec at 95°C; followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The primers used for qPCR were as follows: Bax forward (F), 5′-CAGTTGAAGTTGCCATCAGC-3′ and reverse (R) 5′-ATGCGTCCACCAAGAAGC-3′; Bcl-2 F, 5′-GCGACGAGAGAAGTCATCC-3′ and R, 5′-AGCCTGAGAGCAACCCAAT-3′, STIM1 F, 5′-CCCTTCCAGATCCTTCATCA-3′ and R, 5′-AAGGACTTCATGCTGGTGGT-3′; Orai1 F, 5′-GTGCCCGGTGTTAGAGAATG-3′ and R, 5′-TCCCTGGTCAGCCATAAGAC-3′; and GAPDH F, 5′-AAGAGGGATGCTGCCCTTAC-3′ and R, 5′-TACGGCCAAATCCGTTCACA-3′. The mRNA expression levels of the target genes were normalized to those of GAPDH and fold changes were quantified using the 2−ΔΔCq method (33).
Western blotting
Myocardial tissues were harvested and lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration of the supernatant was determined using a BCA protein assay kit. Equivalent amounts of total protein (30–60 µg protein/lane) were separated via SDS-PAGE on a 8–12% gel. Subsequently, separated protein was transferred to a PVDF membrane (MilliporeSigma). After blocking with 5% non-fat milk at room temperature for 2 h, the membranes were incubated with primary antibodies against STIM1 (1:1,000), Bax (1:1,000), Bcl-2 (1:1,000), or Orai1 (1:500) at 4°C overnight. Following the primary incubation membranes were washed with Tris-buffered saline containing 0.1% Tween-20 for three times and then incubated followed with secondary antibodies (1:5,000; cat. no. BL003A; Biosharp Life Science, Inc.) at room temperature for 2 h. Membranes were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and were semi-quantified using Quantity One v4.6.6 software (Bio-Rad Laboratories, Inc.). GAPDH (cat. no. BS60630; 1:8,000; Bioworld Technology, Inc.) was used as a loading control.
Cell culture and treatment
H9C2 cells were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 mg/ml streptomycin at 37°C with 5% CO2. H9C2 cells were randomly assigned to two groups: i) Control (PBS stimulation); ii) LPS (10 µg/ml). Cells were seeded into plates at an appropriate density (1×105/ml) and incubated for 24 h before incubation with LPS or PBS for 0.5–6.0 h at 37°C and were subsequently harvested for analysis (34).
Immunofluorescence staining
Cells from each treatment group were fixed with 4% paraformaldehyde at 4°C for 1 h and then permeabilized with 0.5% Triton X-100 at room temperature for 10 min. After blocking with 1% BSA at 4°C for 30 min the cells were incubated with anti-STIM1 (1:100) and anti-Orai1 (1:100) primary antibodies at 4°C overnight. Samples were then incubated with dyLightFluor® 488-conjugated donkey anti-rabbit (1:400) or anti-mouse (1:400) IgG secondary antibody for 1 h at room temperature and cells were stained with 10 µg/ml DAPI for 7 min in the dark at room temperature. Representative images were captured using the Olympus BX51 fluorescent microscope (Olympus Corporation).
Store-operated Ca2+ entry in cardiomyocytes
SOCE was quantified as described previously (18). Briefly, H9C2 cells were loaded with 5 µmol/l Fluo-3/AM (excitation at 488 nm) for 40 min at 37°C in normal Tyrode's solution (140 mmol/l NaCl, 5.4 mmol/l KCl, 1 mmol/l MgCl2, 2 mmol/l CaCl2, 5.5 mmol/l glucose and 5 mmol/l HEPES at pH 7.4) in the dark and LPS (10 µg/ml) was administered for 40 min at 37°C. After dye loading, cells were washed with PBS three times within 20 min at room temperature. To prevent store refilling and deletion of intracellular Ca2+ stores, cells were treated with 4 µmol/l TG in Ca2+-free Tyrode's solution. SOCE was triggered by the addition of 1.8 mmol/l Ca2+ to the solution. The cells were captured first before TG or Ca2+ was added. Real-time fluorescent images (all cells) were recored every 2 sec using confocal microscopy (Nikon A1; Nikon Corporation) within 1 h and were analysed using the supporting software (Nikon NIS-Elements AR 4.XX.00). The cells were still alive when the images were recorded, thus H9C2 cells were stimulated by LPS for 1–2 h (40 min dying at 37°C, 20 min washing and 0–1 h recording at room temprature).
Statistical analysis
All experiments were performed in triplicate. Data are presented as the mean ± SD. All analyses were performed using SPSS version 16.0 software (SPSS, Inc.). Statistical significance was assessed using two-tailed unpaired Student's t-test (two groups) and one-way ANOVA followed by Tukey's post hoc test (three or more groups). P<0.05 was considered to indicate a statistically significant difference.
Results
Establishment of septic myocardial depression
Sepsis-induced alterations in cardiac structure and function were analysed according to the survival rate, cardiac contractility and myocardial injury. The survival rate of mice in the sham group was 100% following the CLP operation (Fig. 1A). However, compared with the sham group a marked decrease in survival rate was observed following CLP, with survival rates of 46.7% at 24 h and 20% at 72 h (vs. sham group, P<0.001). Changes in survival rate in the CLP group indicated the success of the septic animal model. Myocardial sections were stained with H&E to assess the damage to the myocardium induced by CLP. Cardiomyocytes were demonstrated to be intact and the cardiac muscle cross striations were clear in the sham group. In the CLP group, cardiomyocytes were destroyed and the cardiac muscle cross striations were no longer clear. Damage to the myocardial tissues by CLP continued following the operation (Fig. 1B). Changes in cardiac function resulting from sepsis were analysed using echocardiography. The results demonstrated significant reductions in the LVEF (10.7%) and LVES (5.7%) in the CLP group at 6 h (P<0.05) compared with the sham 6 h group (Fig. 1C-E). No significant change in the LVEF or LVES was observed at 12 and 24 h in the CLP group compared with the corresponding time-point sham group.
Myocardial apoptosis is induced in septic mice
The myocardial sections damaged by the CLP operation, as confirmed by H&E staining, were used to assess the rate of cardiomyocyte apoptosis induced by sepsis via TUNEL staining. The rate of cardiomyocyte apoptosis was 6% in the 24 h sham group compared with 33.5% at 6 h (P<0.001), 43.1% at 12 h (P<0.001) and 55.3% at 24 h (P<0.001) following CLP, which indicated that the myocardial apoptosis rate significantly increased over time following the operation compared with the sham group (Fig. 2A and B). The mRNA and protein expression levels of apoptosis-related Bax and Bcl-2 were assessed using RT-qPCR and western blotting, respectively, to investigate the activation of sepsis-induced myocardial apoptosis. The ratio of Bax/Bcl-2 mRNA and protein expression levels were significantly increased in the CLP group at 6 (P<0.001) and 12 h (P<0.01) compared with the corresponding sham groups (Fig. 2C-E). The peak Bax/Bcl-2 ratio was observed at 6 h following the CLP operation and both mRNA and protein levels returned to normal at 24 h.
Orai1 glycosylation is increased in septic mice
Myocardium exhibiting apoptotic activation were obtained from septic mice to evaluate changes in STIM1 and Orai1 expression levels via RT-qPCR and western blotting. The results demonstrated that STIM1 and Orai1 mRNA and protein expression levels were not significantly different between the CLP and sham groups at 6, 12, or 24 h (Fig. 3). Interestingly, significant increases in Orai1 glycosylation were observed at 6 h after the CLP operation compared with the sham 6 h group (P<0.001).
LPS increases cytosolic Ca2+ levels and induces SOCE in H9C2 cells
In vitro changes in the cytosolic Ca2+ concentration and SOCE were observed in septic cardiomyocytes. The Ca2+ levels in the ER were depleted by TG in Ca2+-free medium. Ca2+ was re-added to the medium and fluctuations in the cytosolic Ca2+ concentration were recorded using Fluo-3/AM fluorescence (Fig. 4A). The cytosolic Ca2+ concentration initially decreased before levelling off and then abruptly and markedly increased after Ca2+ was added to the medium. SOCE was responsible for the significant gain in fluorescence intensity after Ca2+ was added compared with the control (without TG or Ca2+), as observed in H9C2 cells stimulated with LPS for 1–2 h (P<0.001) (Fig. 4B and C). The cytosolic Ca2+ concentration in H9C2 cells was also significantly enhanced following 1–2 h LPS stimulation compared with the control group (P<0.05) (Fig. 4D).
LPS-induced activation of STIM1 clustering and redistribution in H9C2 cells
Immunofluorescent staining of STIM1 was performed in H9C2 cells following LPS treatment for 30 min, 1, 2, 4 and 6 h. The distribution of STIM1 at 30 min of LPS treatment was the same as that in the control group (equivalent PBS used), although changes in the translocation of STIM1 at the ER-plasma membrane junction occurred at 1 and 2 h following LPS treatment. The distribution of STIM1 returned to normal at 4 and 6 h compared with the control group (Fig. 5A). Immunofluorescent staining of Orai1 was performed following LPS stimulation for 1 and 4 h in H9C2 cells. Immunofluorescent analysis demonstrated that LPS had no effect on the distribution and expression of Orai1 compared with the control (equivalent PBS used) (Fig. 5B).
Discussion
The present study demonstrated that myocardial dysfunction, cardiac injury and myocardial depression may be associated with increased intracellular Ca2+ concentrations caused by STIM1/Orai1-mediated SOCE. Cardiac contractile function was significantly reduced at 6 h and morphological changes in the cardiac tissues and the myocardial apoptosis rate were increased at 6, 12 and 24 h in the CLP compared with the corresponding sham groups. The mRNA and protein expression levels of Bax/Bcl-2 were significantly enhanced at 6 and 12 h and glycosylation of Orai1 was significantly increased at 6 h in the myocardium of septic mice. In H9C2 cells treated with LPS, we detected enhanced intracellular Ca2+ concentration and SOCE at 1 to 2 h and the change of clustering and distribution of STIM1 at 1, 2 h.
Septic myocardial depression occurs during the very early stage of sepsis (35). Although no consistent diagnostic criteria exist for septic myocardial dysfunction, it is clearly characterized by reduced ventricular contractility (36). Changes in ventricular contractility have been demonstrated in patients with sepsis, as well as in in vitro and in vivo experimental studies (37–39). Moreover, myocardium dystfunction in sepsis is reported to be reversable (40). Echocardiography is an irreplaceable tool for the evaluation of septic myocardial dysfunction. LVEF and LVFS are two major parameters used to assess changes in ventricular contractility. In the present study, LVEF and LVFS were reduced at 6 h following the CLP operation, before being restored at 12 h, which indicates ventricular contraction dysfunction during the early stages of sepsis and reversal of septic myocardial dysfunction. These results are in accordance with previous studies (41) and they have demonstrated the success of this animal model of septic myocardial dysfunction.
Apoptosis is a type of programmed cell death that has been demonstrated to contribute to septic myocardial depression (9). Bax and Bcl-2 belong to the Bcl-2 family of proteins associated with apoptosis and changes in these proteins over time have been observed in the heart tissue of septic animal models (41). Bcl-2 overexpression may prevent myocardial dysfunction and cardiac apoptosis in rodent models of sepsis (42). Furthermore, certain drugs and reagents have a protective effect on septic myocardial depression by inhibiting apoptosis (43–45). In the present study, the occurrence of myocardial apoptosis in sepsis was detected using TUNEL staining. The time-dependent expression (Bax/Bcl-2 was first elevated, reaching to peak, then decreased) of Bax and Bcl-2 occurred at both the mRNA and protein expression levels, which is consistent with other studies (41,42). These results further demonstrated the success of this animal model of septic myocardial injury.
The mechanisms underlying septic myocardial depression include mitochondrial dysfunction, inflammation, excessive formation of reactive oxygen species and nitric oxide, an imbalance in Ca2+ homeostasis and regulation of the G protein receptor kinase (46–48). In a Previous study has shown that short-term (2 min) stimulation of neonatal cardiomyocytes with septic serum was demonstrated to be sufficient to increase the basal Ca2+ concentration and reduce the amplitude of transient Ca2+ concentrations (8). An increase in the basal Ca2+ concentration was still detected at 24 h following stimulation; however, a reduction in the transient Ca2+ amplitude was not observed at this timepoint (8). These previous findings indicated that cardiac dysfunction is reversible, whereas myocardial cellular apoptosis is irreversible. Similarly, short-term (3 h) treatment of adult ventricular cardiomyocytes with LPS increases the mean cytoplasmic Ca2+ concentration (49). In accordance with other studies reporting an increase in the basal Ca2+ concentration after LPS treatment in cardiomyocytes, a significant elevation in the mean cytosolic Ca2+ concentration was observed in H9C2 cells stimulated with LPS for 1–2 h.
Ca2+ homeostasis is regulated by Ca2+ channels, such as LTCCs, Na+/Ca2+ exchange channels, Orai1 and transient receptor potential canonical channels, located in the plasma membrane, as well as the ryanodine receptor and SR Ca2+ ATPase located in the ER membrane (29,50–52). Ca2+ transporters are triggered by various stimuli, including voltage changes, Ca2+ release and depletion of ER (53). Changes in the Ca2+ level in the plasma membrane may contribute to normal physiological functions, such as the contraction/relaxation cycle, the basal signalling pathway and various pathological states, such as cell structural damage, apoptosis and cell hypertrophy. Among the Ca2+ transporters contributing to Ca2+ entry, LTCCs function in transient cytoplasm Ca2+ cycling of the cardiomyocyte contraction/relaxation cycle. SOCE is part of the signalling pathway responsible for cell survival and death and is associated with sustained elevation of Ca2+ levels in the cytoplasm (18,19). The results of the present study indicated that the Ca2+ levels rise between 1 and 2 h in the cytoplasm, which suggested that SOCE may contribute to the occurrence and development of septic myocardial injury.
SOCE is triggered by the depletion of Ca2+ in the ER, which may be sensed by STIM1 and Ca2+ entry is activated by Ca2+ channels, such as Orai1 (17,54). STIM1 and Orai1 are expressed in the heart; however, their expression levels and SOCE activation are lower in adult cardiomyocytes compared with embryonic or new-born cardiomyocytes (18). This may limit investigations of STIM1/Orai1-mediated SOCE in cardiomyocytes. STIM1/Orai1-mediated SOCE was initially discovered in non-excitable cells and was thought to be absent in excitable cells (55). STIM1/Orai1-mediated SOCE was later discovered in excitable cells and was reported to contribute to hyperplasia of smooth muscle cells and hypertrophy of cardiomyocytes (20,56). However, to the best of our knowledge the role of STIM1/Orai1-mediated SOCE in myocardial injury has seldom been reported and the effect of STIM1/Orai1-mediated SOCE in septic myocardial depression has never been reported. The present study demonstrated significantly increased SOCE in cardiomyocytes stimulated by LPS, which indicated a potential relationship between SOCE and septic myocardial depression.
Slight changes in the SOCE-related proteins STIM1 and Orai1 were also observed in the present study, which differs from increased expression of STIM1 and Orai1 previously been found in the model of cardiac hypertrophy (19,57). The redistribution of STIM1 to the plasma membrane was observed for a short period following LPS stimulation and was time-dependent. Translocation of STIM1 was detected by immunofluorescence and the results indicated the oligomerized aggregation of STIM1 at the ER-plasma membrane junction. Moreover, a previous study proposed that short-term TG stimulation results in changes in the clustering and redistribution of STIM1, but not in the expression levels (19). Furthermore, two STIM1 subtypes have been reported, with STIM1L (a long splice variant of STIM1) exhibiting more prominent changes compared with STIM1 in cardiac hypertrophy models (18). In the present study two bands representing STIM1 were detected via western blotting, which corresponded to STIM1L and STIM1. However, no significant difference in the protein expression level of either STIM1L and STIM1were observed in the present study.
No change in Orai1 expression level was observed by qPCR or western blot analysis in our experiments, while a change was observed in the myocardial hypertrophy model. The plasma membrane protein Orai1 was observed at 32 kDa in most protein expression experiments. However, the SOCE rate was dependent on glycosylation of Orai1, which produced a 56 kDa marker in fibroblasts and T cells (58). In the present study the protein expression of Orai1 at both 32 and 56 kDa was detected and significant changes in Orai1 glycosylation (56 kDa) were demonstrated in the CLP group. Orai1 glycosylation was significantly increased at 6 h and returned to the baseline, which was accordance with the change in sepsis-induced cardiac dysfunction and myocardial apoptosis. Although altered expression levels of STIM1 and Orai1 were not observed in the present study, the translocation of STIM1 and significantly increased glycosylation of Orai1 indicated a potential association between STIM1/Orai1-mediated SOCE and septic myocardial depression.
In summary, the present study demonstrated that STIM1/Orai1-mediated SOCE may be associated with septic myocardial depression. Therefore, the regulation of STIM1/Orai1-mediated SOCE may have the potential to alleviate septic myocardial depression. However, further studies are needed to to confirm the exact role of STIM1/Orai1-mediated SOCE in septic cardiac depression and to determine its clinical effect and toxicity.
Acknowledgements
Not applicable.
Funding
The present study was funded by the Zhejiang Provincial Natural Science Foundation (grant no. LQ15H150003), and in part, by grants from the National Natural Science Foundation of China (grant no. 81772112 and 81871583).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
JY, ML GZ and ZL conceived and designed the study. JY, XH, QL, ZJ, SZ and GH performed the experiments and analysed the data. JY, GZ and ZL wrote the manuscript. JY, GZ, GH and ZL confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The experiments involving animals were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Wenzhou Medical University (approval no. wydw2016-0217; Wenzhou, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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